HPAC cells were
seeded in U-bottom 96 well-plates pre-treated with 0.5% polyHEMA [poly(2-hydroxyethyl
methacrylate), Sigma in 95% ethanol] at 2 × 104 cells
per well. The plate was then centrifuged at 700 rpm at 37 °C
using a BMG Labtech ClarioStar (plus) plate reader for 10 min. The
cells were incubated for 96 h to allow spheroid formation before any
dye was added for imaging studies. Ru-bqp-ester, Ru-bqp-MPP, and Ru-bqp-R8
were added to the spheroids at 30 and 100 μM in the 96 well
plates and after 24 h incubation, the spheroids were carefully transferred
to an 8-chamber slide (ibidi), with a single spheroid per chamber,
and directly imaged using a Leica TCS DMi8 confocal microscope (40×
oil immersion objective). Hoechst 33342 nuclear stain (1 μg/mL)
was added to the spheroids for 45 min as a contrast agent and excited
using a 405 nm laser with emission collected between 425 and 475 nm.
The Ru(II) complexes were excited using a white light laser at 490
nm and emission collected between 580 and 730 nm. Spheroid images
were acquired using z-scanning across the z-axis
of the samples. On average, 40–50 images were acquired per
z-scan and used to obtain 3D spheroid reconstructions using Leica
Application Suite X (LAS X) software.
seeded in U-bottom 96 well-plates pre-treated with 0.5% polyHEMA [poly(2-hydroxyethyl
methacrylate), Sigma in 95% ethanol] at 2 × 104 cells
per well. The plate was then centrifuged at 700 rpm at 37 °C
using a BMG Labtech ClarioStar (plus) plate reader for 10 min. The
cells were incubated for 96 h to allow spheroid formation before any
dye was added for imaging studies. Ru-bqp-ester, Ru-bqp-MPP, and Ru-bqp-R8
were added to the spheroids at 30 and 100 μM in the 96 well
plates and after 24 h incubation, the spheroids were carefully transferred
to an 8-chamber slide (ibidi), with a single spheroid per chamber,
and directly imaged using a Leica TCS DMi8 confocal microscope (40×
oil immersion objective). Hoechst 33342 nuclear stain (1 μg/mL)
was added to the spheroids for 45 min as a contrast agent and excited
using a 405 nm laser with emission collected between 425 and 475 nm.
The Ru(II) complexes were excited using a white light laser at 490
nm and emission collected between 580 and 730 nm. Spheroid images
were acquired using z-scanning across the z-axis
of the samples. On average, 40–50 images were acquired per
z-scan and used to obtain 3D spheroid reconstructions using Leica
Application Suite X (LAS X) software.